Synthesis of biologically active compounds in cells

ABSTRACT

This invention relates to a new method of synthesis of biologically active substances of determined structure directly in the cells of living organisms containing specific RNA or DNA molecules of determined sequence. The method is based on the hybridization of two or more oligomers bound with biologically inactive precursors of biologically active substances to specific RNA or DNA in vivo in the cells of living organisms. After hybridization of the oligomers to RNA or DNA the biologically inactive precursors bound to the 5′ and/or 3′ ends of the oligomers can interact with each other to make biologically active form of the substances. This changing of properties is due to chemical reactions which bind the biologically inactive precursors through a chemical bond into a biologically active form of the whole compound.

BACKGROUND ART

[0001] The use of oligo(ribo)nucleotides and their analogues asanticancer and antiviruses theraupetic agents was first proposed severalyears ago. (Uhlmann, 1990) The great number of different modificationsof the oligonucleotides and the methods of their use have since beendeveloped.

[0002] Two basic interactions between oligonucleotides and nucleic acidsare known (Moser and Dervan, 1987)

[0003] 1. Watson-Crick base pairing (Duplex structure)

[0004] 2. Hoogsten base pairing (Triplex structure)

[0005] Oligonucleotides can form duplex and/or triplex structures withDNA or RNA of cells and so regulate transcription or translation ofgenes.

[0006] It has been proposed that different substances which can cleavetarget nucleic acids or inhibit important cellular enzymes could becoupled to oligomers. The use of such conjugates as therapeutic agentshas been described.(U.S. Pat. Nos., 5,177,198; 5,652,350).

[0007] Other methods are based on the coupling of different biologicallyactive substances, such as toxins, to monoclonal antibodies which canthen recognise receptors or other structures of cancer cells, or cellsinfected with viruses. Monoclonal antibodies can then specificallyrecognise cancer cells and in this way transport toxins to these cells.But these methods are inefficient due to the high level of nonspecificinteractions between antibodies and other cells, which leads to delivaryof the toxins or other biologically active compounds to the wrong cells.

[0008] In 1979 I. M. Klotz and co-authors proposed a method forcomplementary carrier peptide synthesis based on a template-directlyedscheme (J. A. Walder et al. 1979) The method proposed the synthesis ofpeptides on a solid support using unprotected amino acids, and thesubsequent hybridization of oligonucleotides on the template. Thismethod was established only for synthesis of peptides in vitro usingsolid supports of a different origin, and involved many synthesis stepsto obtain peptides of the determined structure.

[0009] M. Masuko and co-authors proposed another method for in vitrodetection of specific nucleic acids by excimer formation from twopyrene-labeled probes (Ebata, K. et al. 1995).

[0010] My invention allows the synthesis of different BACs of determinedstructure directly in living organisms only in cells which have specificRNA or DNA sequences. In this way, BACs will be delivered only to thosecells where specific nucleic acids are produced.

DISCLOSURE OF INVENTION Definitions

[0011] “Nulceomonomer”

[0012] The term “nucleomonomer” means a “Base” chemically bound to “S”moieties. Nucleomonomers can include nucleotides and nucleosides such asthymine, cytosine, adenine, guanine, diaminopurine, xanthine,hypoxanthine, inosine and uracil. Nucleomonomers can bind each other toform oligomers which can be specifically hybridised to nucleic acids ina sequence and direction specific manner.

[0013] The “S” moieties used herein include D-ribose and2′-deoxy-D-ribose. Sugar moieties can be modified so that hydroxylgroups are replaced with a heteroatom, aliphatic group, halogen, ethers,amines, mercapto, thioethers and other groups. The pentose moiety can bereplaced by a cyclopentane ring, a hexose, a 6-member morpholino ring;it can be aminoacids analogues coupled to base, bicyclic riboacetalanalogues, morpholino carbamates, alkanes, ethers, amines, amides,thioethers, formacetals, ketones, carbamates, ureas, hydroxylamines,sulfamates, sulfamides, sulfones, glycinyl amides other analogues whichcan replace sugar moieties. Oligomers obtained from the mononucleomerscan form stabile duplex and triplex structures with nucleic acids.(Nielsen P. E. 1995, U.S. Pat. No. 5,594,121).

[0014] “Base”

[0015] “Base” (designated as “Ba”) includes natural and modified.purines and pyrimidines such as thymine, cytosine, adenine, guanine,diaminopurine, xanthine, hypoxanthine, inosine, uracil, 2-aminopyridine,4,4-ethanocytosine, 5-methylcytosine, 5-methyluracil, 2-aminopyridineand 8-oxo-N(6)-methyladenine and their analogues. These may include, butare not limited to adding substituents such as —OH, —SH, —SCH(3),—OCH(3), —F, —Cl, —Br, —NH(2), alkyl, groups and others. Also,heterocycles such as triazines are included.

[0016] “Nucleotide”

[0017] Nucleotide as used herein means a base chemically bound to asugar or sugar analogues having a phosphate group or phosphate analog.

[0018] “Oligomer”

[0019] Oligomer means that at least two “nucleomonomers” (defined above)are chemically bound to each other. Oligomers can beoligodeoxyribonucleotides consisting of from 2 to 200 nucleotides,oligoribonucleotides consisting of from 2 to 200 nucleotides, ormixtures of oligodeoxyribonucleotides and oligoribonucleotides. Thenucleomonomers can bind each other through phosphodiester groups,phosphorothioate, phosphorodithioate, alkylphosphonate,boranophosphates, acetals, phosphoroamidate, bicyclic riboacetalanalogues morpholino carbamates, alkanes, ethers, amines, amides,thioethers, formacetals, ketones, carbamates, ureas, hydroxylamines,sulfamates, sulfamides, sulfones, glycinyl amides and other analogueswhich can replace phosphodiester moiety. Oligomers are composed ofmononucleomers or nucleotides. Oligomers can form stable duplexstructures via Watson-Crick base pairing with specific sequences of DNA,RNA, mRNA, rRNA and tRNA in vivo in the cells of living organisms orthey can form stable triplex structures with double stranded DNA ordsRNA in vivo in the cells of living organisms.

[0020] “Alkyl”

[0021] “Alkyl” as used herein is a straight or branched saturated grouphaving from 1 to 10 carbon atoms. Examples include methyl, ethyl,propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl and thelike.

[0022] “Alkenyl”

[0023] “Alkenyl” as used herein is a straight- or branched-chainolefinically-unsaturated group having from two to 25 carbon atoms. Thegroups contain from one to three double bounds. Examples include vinyl(—CHdbdCH(2), 1-propenyl (—CHdbdCH—CH(3)), 2-methyl-1-propenyl(—CHdbdC(CH(3))—CH(3)) and the like

[0024] “Alkynyl”

[0025] “Alkynyl” as used herein is a straight or branchedacetynically-unsaturated groups having from two to 25 carbon atoms. Thegroups contain from one to three triple bounds. Examples include1-alkynyl groups include ethynyl (—CtbdCH), 1-propynyl (—CtbdC—CH(3)),1-butynyl (—CtbdC—CH(2 —CH(3)), 3-methyl-butynyl (—CtbdC—CH(CH(3))—CH(3)), 3,3-dimethyl-butynyl (—CtbdC—C(CH(3))(3)), 1-pentynyl(—CtbdC—CH(2, —CH(2 —CH(3)) and 1,3-pentadiyiyl (—CtbdC—CtbdC—CH(3)) andthe like.

[0026] “Aryl”

[0027] “Aryl” as used herein includes aromatic groups having from 4 to10 carbon atoms. Examples include phenyl, naphtyl and like this.

[0028] “Heteroalkyl”

[0029] “Heteroalkyl” as used herein is an alkyl group in which 1 to 8carbon atoms are replaced with N (nitrogen), S (sulfur) or O (oxygen)atoms. At any carbon atom there can be one to three substituents. Thesubstituents are selected from: —OH, —SH, —SCH₃, —OCH₃, halogen, —NH₂,—NO₂, —S(O)—, —S(O)(O)—, —O—S(O)(O)—O—, —O—P(O)(O)—O—, —NHR. Here R isalkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic, carbocyclic andlike this groups.

[0030] “Heteroalkenyl”

[0031] “Heteroalkenyl” as used herein is an alkenyl group in which 1 to8 carbon atoms are replaced with N (nitrogen), S (sulfur) or O (oxygen)atoms. At any carbon atom there can be one to three substituents. Thesubstituents are selected from group —OH, —SH, —SCH₃, —OCH₃, halogen,—NH₂, NO₂, —S(O)—, —S(O)(O)—, —O—S(O)(O)—O—, —O—P(O)(O)—O—, —NHR. Here Ris alkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic, carbocyclicand like this groups.

[0032] “Heteroalkynyl”

[0033] “Heteroalkynyl” as used herein is an alkynyl group in which 1 to8 carbon atoms are replaced with N (nitrogen), S (sulfur) or O (oxygen)atoms. At any carbon atom there can be one to three substituents. Thesubstituents are selected from group —OH, —SH, —SCH₃, —OCH₃, halogen,—NH₂, NO₂, —S(O)—, —S(O)(O)′—, —O—S(O)(O)—O—, —O—P(O)(O)—O—, —NHR. HereR is alkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic,carbocyclic and like this groups.

[0034] “Heteroaryl”

[0035] “Heteroaryl” as used herein means an aromatic radicals comprisingfrom 5 to 10 carbon atoms and additionally containing from and to threeheteroatoms in the ring selected from group S, O or N. The examplesinclude but not limited to: furyl, pyrrolyl, imidazolyl, pyridylindolyl, quinolyl, benzyl and the like. One to three carbon atoms ofaromatic group can have substituents selected from —OH, —SH, —SCH₃,—OCH₃, halogen, —NH₂, NO₂, —S(O)—, —S(O)(O)—, —O—S(O)(O)—O—,—O—P(O)(O)—O—, —NHR, alkyl group. Here R is alkyl, alkenyl, aryl,heteroaryl, alkynyl, heterocyclic, carbocyclic or similar groups.

[0036] “Cycloheteroaryl”

[0037] “Cycloheteroaryl” as used herein means a group comprising from 5to 25 carbon atoms from one to three aromatic groups which are combinedvia a carbocyclic or heterocyclic ring. An illustrative radical isfluorenylmethyl. One to two atoms in the ring of aromatic groups can beheteroatoms selected from N, O or S. Any carbon atom of the group canhave substituents selected from —OH, —SH, —SCH₃, —OCH₃, halogen, —NH₂,NO₂, —S(O)—, —S(O)(O)—, —O—S(O)(O)—O—, —O—P(O)(O)—O—, —NHR, alkyl group.Here R is alkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic andcarbocyclic and like this groups.

[0038] “Carbocyclic”

[0039] “Carbocyclic” as used herein designates a saturated orunsaturated ring comprising from 4 to 8 ring carbon atoms. Carbocyclicrings or groups include cyclopentyl, cyclohexyl and phenyl groups. Anycarbon atom of the group can have substituents selected from —OH, —SH,—SCH₃, —OCH₃, halogen, —NH₂, NO₂, —S(O)—, —S(O)(O)—, —O—S(O)(O)—O—,—O—P(O)(O)—O—, —NHR, alkyl group. Here R is alkyl, alkenyl, aryl,heteroaryl, alkynyl, heterocyclic and carbocyclic and like this groups.

[0040] “Heterocyclic Ring”

[0041] “Heterocyclic ring” as used herein is a saturated or unsaturatedring comprising from 3 to 8 ring atoms. Ring atoms include C atoms andfrom one to three N, O or S atoms. Examples include pyrimidinyl,pyrrolinyl, pyridinyl and morpholinyl. At any ring carbon atom there canbe substituents such as —OH, —SH, —SCH₃, —OCH₃, halogen, —NH₂, NO₂,—S(O)—, —S(O)(O)—, —O—S(O)(O)—O—, —O—P(O)(O)—O—, —NHR, alkyl. Where R isalkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic and carbocyclicand like this groups.

[0042] “Hybridization”

[0043] “Hybridization” as used herein means the formation of duplex ortriplex structures between oligomers and ssRNA, ssDNA, dsRNA or dsDNAmolecules. Duplex structures are based on Watson-Crick base pairing.Triplex structures are formed through Hoogsteen base interactions.Triplex structures can be parallel and antiparallel.

[0044] The word “halogen” means an atom selected from the groupconsisting of F (fluorine), Cl (clorine), Br (bromine) and I (iodine)

[0045] The word “hydroxyl” means an—OH group:

[0046] The word “carboxyl” means an—COOH function.

[0047] The word “mercapto” means an—SH function.

[0048] The word “amino” means—NH(2) or—NHR. Where R is alkyl, alkenyl,aryl, heteroaryl, heteroalkyl, alkynyl, heterocyclic, carbocyclic andlike this groups.

[0049] “Biologically Active Compounds (BACs)”

[0050] “Biologically active compound as defined herein include but arenot limited to:

[0051] 1) biologically active peptides and proteins consisting ofnatural aminoacids and their synthetic analogues L, D, or DLconfiguration at the alpha carbon atom selected from valine, leucine,alanine, glycine, tyrosine, tryptophan, tryptophan isoleucine, proline,histidine, lysin, glutamic acid, methionine, serine, cysteine, glutaminephenylalanine, methionine sulfoxide, threonine, arginine, aspartic acid,asparagin, phenylglycine, norleucine, norvaline, alpha-aminobutyricacid, O-methylserine, O-ethylserine, S-methylcysteine, S-benzylcysteine,S-ethylcysteine, 5,5,5-trifluoroleucine and hexafluoroleucine. Alsoincluded are other modifications of aminoacids which include but are notlimited to, adding substituents at carbon atoms such as —OH, —SH, —SCH₃,—OCH₃, —F, —Cl, —Br, —NH₂. The peptides can be also glycosylated andphosphorylated.

[0052] 2) Cellular proteins which include but are not limited to:enzymes, DNA polymerases, RNA polymerases, esterases, lipases,proteases, kinases, transferases, transcription factors, transmembraneproteins, membrane proteins, cyclins, cytoplasmic proteins, nuclearproteins, toxins and like this.

[0053] 3) Biologically active RNA such as mRNA, ssRNA, rsRNA and likethis.

[0054] 4) Biologically active alkaloids and their synthetic analogueswith added substituents at carbon atoms such as —OH, —SH, —SCH₃, —OCH₃,—F, —Cl, —Br, —NH₂, alkyl straight and branched.

[0055] 5) Natural and synthetic organic compounds which can be:

[0056] a) inhibitors and activators of the cellular metabolism;

[0057] b) cytolitical toxins;

[0058] c) neurotoxins;

[0059] d) cofactors for cellular enzymes;

[0060] e) toxins;

[0061] f) inhibitors of the cellular enzymes.

[0062] “Precursor(s) of Biologically Active Substances (PBAC(s))”

[0063] “Precursors of biologically active compounds (PBACs)” as usedherein are biologically inactive precursors of BACs which can form wholeBACs when bound to each other through chemical moiety(ies) “m” orsimultaneously through chemical moieties “m” and “m^ 1”. “m” and “m^ 1”are selected independently from: —S—S—, —O—, —NH—C(O)—, —C(O)—NH—,—C(O)—, —NH—, dbdN—, —C(O)O—, —C(O)S—, —S—, —C(S)S—, —C(S)O—, —N═N—.

[0064] Biologically active peptides and proteins are synthesized fromshorter biologically inactive peptides. These shorter peptides as usedherein are also biologically inactive precursors of biologically activecompounds.

[0065] Biologically active RNAs can be synthesized from biologicallyinactive oligoribonucleotides.

[0066] “Oligomer-PBAC”

[0067] “Oligomer-PBAC” as used herein means a precursor of a BAC (PBAC)which is chemically bound at the first and/or last mononucleomer at the3′ and/or 5′ ends of the oligomer through the chemical moieties L^ 1and/or L{circumflex over (2)}. Chemical moieties L^ 1 and L{circumflexover (2)} can be bound directly to a base or to a sugar moiety or tosugar moiety analogues or to phosphates or to phosphate analogues,

[0068] “Oligomer_(n)-PA_(n)”

[0069] “Oligomer_(n)-PA_(n)” as used herein means the precursor of abiologically active protein or RNA which is chemically bound at thefirst and/or last mononucleomer at the 3′ and/or 5′ ends of the oligomerthrough the chemical moieties L^ 1 and/or L^ 2. n means the ordinalnumber of the oligomer of PA. PAs are biologically inactive peptides orbiologically inactive oligoribonucleotides. Wherein n is selected from 2to 300.

[0070] a) In Formulas 1 to 4 PBACs are designated as “A” and “B”

[0071] A-m-B is equal to a whole BAC “T”

[0072] “m” is selected independently from —S—S—, —O—, —NH—C(O)—,—C(O)—NH—, —C(O)—, —NH—, dbdN—, —C(O)O—, —C(O)S—, —S—, —C(S)S—, —C(S)O,—N═N—.

[0073] A-O-B is equal to a whole BAC “T”

[0074] A-NH—C(O)-B is equal to a whole BAC “T”

[0075] A-C(O)—NH-B is equal to a whole BAC “T”

[0076] A-C(O)-B is equal to a whole BAC “T”

[0077] A-NH-B is equal to a whole BAC “T”

[0078] A-dbdN-B is equal to a whole BAC “T”

[0079] A-C(O)O-B is equal to a whole BAC “T”

[0080] A-C(O)S-B is equal to a whole BAC “T”

[0081] A-C(S)S-B is equal to a whole BAC “T”

[0082] A-S—S-B is equal to a whole BAC “T”

[0083] A-C(S)O-B is equal to a whole BAC “T”

[0084] A-N═N-B is equal to a whole BAC “T”

[0085] b) Biologically active compounds can be formed through moieties“m” and “m^ 1”. “m” and “m{circumflex over (1)}” are selectedindependently from: —S—S—, —O—, —NH—C(O)—, —C(O)—NH—, —C(O)—, —NH—,dbdN—, —C(O)O—, —C(O)S—, —S—, —C(S)S—, —C(S)O, —N═N—, so that

[0086] is equal to biologically active compound “T” a BAC is representedon figure

[0087] c) In Formulas 5 to 7, precursors of BACs (PBACs) are designatedas “PA_(n)”, where n is selected from 2 to 300. “PA” are peptidesconsisting of from 2 to 100 amino acids or oligoribonucleotidesconsisting of from 2 to 50 ribonucleotides.

[0088] {PA₁-m-PA₂-m-PA₃-m- . . .-m-PA_(n-3)-m-PA_(n-2)-m-PA_(n-1)-m-PA_(n)} is equal to BAC. BACs inthis case are proteins or RNAs. Proteins can be enzymes, transcriptionfactors, ligands, signaling proteins, transmembrane proteins,cytolitical toxins, toxins, cytoplasmic proteins, nuclear proteins andthe like.

DETAILED DISCLOSURE OF THE INVENTION

[0089] This invention relates to the synthesis of biologically activecompounds directly in the cells of living organisms. This is achieved bythe hybridization of two or more oligomers to cellular RNA or DNA. Theseoligomers are bound to biologically inactive PBACs (oliogmer-PBACs)containing chemically active groups.

[0090] BAC can be synthesized only in those cells of living organismswhich have specific RNA or DNA molecules of a determined sequence.

[0091] The principle Formulas of the invention are represented below:

[0092] After hybridization of the “Oligomer-PBACs” “A” and “B” tocellular RNA, DNA or dsDNA, the chemically active groups K^ 1 and K^ 2of the oligomer-PBACs “A” and “B” interact with each other to form thechemical moiety “m”, which combines PBACs “A” and “B” into one activemolecule of biologically active compound “T”. The degradation of theoligomers and/or linking moieties L^ 1 and L^ 2 by cellular enzymes orhydrolysis leads to the release of the synthesized BAC “T” directly intothe targeted cells. After hybridization of the oligomer-PBACs tocellular RNA or DNA the distance between the 3′ or 5′ ends of theoligomer A and 5′ or 3′ ends of the oligomer B is from 0 to 7nucleotides of cellular RNA, DNA or dsDNA.

[0093] After hybridization of the “oligomer-PBACs” “A” and “B” tocellular RNA, DNA or dsDNA the chemically active group K^ 2 of theoligomer-PBAC “B” interacts with the linking moiety L^ 1 of theoligomer-PBAC “A” to combine the PBACs through the chemical moiety “m”into one active molecule of biologically active compound “T” with thesubsequent release of one PBAC “B” from the oligomer. The degradation ofthe oligomer and/or linking moieties L^ 1 by cellular enzymes orhydrolysis leads to the release of synthesized BAC “T” directly into thetargeted cells.

[0094] The chemically active group K^ 1 of the oligomer-PBAC A interactswith the linking moiety L^ 2 to combine the PBACs through the chemicalmoiety “m” into one active molecule of the biologically active compound“T” with the subsequnet release of one PBAC “B” from oligomer “B” andthe activation of the chemical moiety L^ 2. After activation, L^ 2interacts with the linking moiety L^ 1 to release the biologicalcompound “T” from the oligomer directly into targeted cells.

[0095] After hybridization, of the “oligomer-PBACs” “A” and “B” tocellular RNA, DNA or dsDNA, the chemically active group K^ 2 of theoligomer-PBAC “B” interacts with the linking moiety L^ 1 of theoligomer-PBAC “A” to combine the PBACs through the chemical moiety “m”.At the same time the chemically active group K^ 1 of the oligomer-PBAC“A” interacts with the linking moiety L^ 2 of the oligomer-PBAc “B” toform chemical moiety m^ 1. Which together with chemical moiety mcombines two “Oligomer-PBACs” into one active molecule of biologicallyactive compound “T”, with the release of BAC from the oligomer.

[0096] After simultaneous hybridization of “Oligomer_(n-1)-PA_(n-1)” and“Oligomer_(n)-PA_(n)” to cellular RNA or DNA, the chemically activegroups K^ 1 and K^ 2 interact with each other to form the chemicalmoiety “m” between “Oligomer_(n-1)-PA_(n-1)” and “Oligomer_(n)-PA_(n)”correspondingly; This step is repeated in the cells n-1 times andcombines n-1 times all “PA_(n)”s into one active molecule of thebiologically active compound “PR” which consists of n PA_(n) so thatcompound {“PA”₁-m-“PA”₂-m-“PA”₃-m-“PA”₄-m- . . .-m-“PA_(n-3)”-m-“PA_(n-2)”-m-“PA_(n-1)”-m-“PA_(n)”} is biologicallyactive compound “PR”. The degradation of the oligomers and/or linkingmoieties L^ 1 and L^ 2 leads to the release of the synthesized BAC “PR”directly into targeted cells of living organism. Here, n is selectedfrom 2 to 2000;

[0097] After simultaneous hybridization of “oligomer_(n-1)-PA_(n-1)” and“oligomer_(n)-PA_(n)” to cellular RNA, DNA or dsDNA, the chemicallyactive group K^ 1 of “oligomer_(n)-PA_(n)” interacts with the linkingmoiety L^ 2 of “oligomer_(n-1)-PA_(n-1)” to bind PA_(n-1) and PA_(n)through chemical moiety “m”. This step is repeated in the cells n-1times and combines n-1 times all PA_(n)s after hybridization of all n“oligomer-PA_(n)”s into one active molecule of the biologically activecompound “PR”, which consists of n PAs so that compound{PA₁-m-PA₂-m-PA₃-m-PA₄-m- . . .-m-PA_(n-3)-m-PA_(n-2)-m-PA_(n)-₁-m-PA_(n)} is equal to the biologicallyactive compound PR. The degradation of the oligomers and/or linkingmoieties L^ 1 by cellular enzymes or hydrolysis leads to the release ofthe synthesized BAC PR directly into targeted cells of living organism,here n is selected from to 2000;

[0098] After simultaneous hybridization of “Oligomer_(n-1)-PA_(n-1)” and“oligomer_(n)-PA_(n)” to cellular RNA, DNA or dsDNA, the chemicallyactive group K^ 1 of “oligomer_(n-1)-PA_(n-1)” interacts with thelinking moiety L^ 2 of “oligomer_(n)-PA_(n)” to bind PA_(n-1) and PA_(n)through chemical moiety “m”. After interaction of K^ 1 with L^ 2, L^ 2is chemically activated so that it can interact with linking moiety L^ 1of the oligomer-PA_(n-1), thus destroying the binding of theoligomer_(n-1) to PA_(n-1). This process is repeated n-1 times, so thatonly whole BAC “PR” comprising from n PA_(n)s {PA₁-m-PA₂-m-PA₃-m-PA₄-m-. . . -m-PA_(n-3)-m-PA_(n-2)-m-PA_(n-1)-m-PA_(n)} is released directlyinto the targeted cells of living organisms, here n is selected from 2to 2000.

[0099] The chemical moieties in the Formulas 1,2,3,4,5,6 and 7 are asfollows:

[0100] m is selected independently from: —S—S—, —N(H)C(O)—, —C(O)N(H)—,—C(S)—O—, —C(S)—S—, —O—, —N═N—, —C(S)—, —C(O)—O—, —NH—, —S—;

[0101] K^ 1 is selected independently from: —NH(2), dbdNH, —OH, —SH, —F,—Cl, —Br, —I, —R^ 1-C(X)—X^ —R^ 2;

[0102] K^ 2 is selected independently from: —NH(2), -dbd-NH, —OH, —SH,—R^ 1C(X)—X^ 1-R^ 2, —F, —Cl, —Br, —I;

[0103] L^ 1 is independently: chemical bond, —R^ 1-, -R^ 1-O—S—R-^ 2-,—R^ 1-S—O—R-^ 2-, —R^ 1-S—S—R^ 2-, —R^ 1S—N(H)—R^ 2-, —R^ 1-N(H)—S—R^2-, —R^ 1-O—N(H)—R^ 2-, —R^ 1-N(H)—O—R^ 2-, —R^ 1-C(X)—X—R^ 2-;

[0104] L^ 2 is independently: chemical bond, —R^ 1, —R^ 1-O—S—R^ 2-, —R^1-S—O—R^ 2-, —R^ 1-S—S—R^ 2-, —R^ 1-S—N(H)—R^ 2-, —R^ 1-N(H)—S—R^ 2-,—R^ 1-O—N(H)—R^ 2-, —R^ 1-N(H)—O—R^ 2-, —R^ 1-C(X)—X^ 1-R^ 2-, —R^1-X—C(X)—X—C(X)—X—R^ 2-;

[0105] R-^ 1 is independently: chemical bond, alkyl, alkenyl, alkynyl,aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl,cycloheteroaryl, carbocyclic, heterocyclic ring, X^ 1-P(X)(X)—X^ 1,—S(O)—, —S(O)(O)—, —X^ 1-S(X)(X)—X^ 1-, —C(O)—, —N(H)—, —N═N—, —X^1P(X)(X)—X^ 1-, —X^ 1-P(X)(X)—X^ 1-.

[0106] P(X)(X)—X^ 1, —X^ 1-P(X)(X)—X^ 1-P(X)(X)—X^ 1P(X)(X)—X^ 1,—C(S)—, any suitable linking group;

[0107] R^ 2 is independently chemical bond, alkyl, alkenyl, alkynyl,aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl,cycloheteroaryl, carbocyclic, heterocyclic ring, X^ 1-P(X)(X)—X^ 1,—S(O)—, —S(O)(O)—, —X^ 1-S(X)(X)—X-^ 1-, —C(O)—, —N(H)—, —N═N—, —X^1-P(X)(X)—X^ 1-, —X^ 1-P(X)(X)—X^ 1-P(X)(X)—X^ 1, —X^ 1-P(X)(X)—X^1-P(X)(X)—X^ 1-P(X)(X)—X^ 1, —C(S)—, any suitable linking group;

[0108] X is independently S, O, NH, Se, alkyl, alkenyl, alkynyl;

[0109] X^ 1 is independently S, O, NH, Se, alkyl, alkenyl, alkynyl.

[0110] In Formulas 1,2,3,4,5,6 and 7 the linking moieties L^ 1 and L^ 2are bound to the first and/or last mononucleomers of the oligomers attheir sugar or phosphate moiety, or directly to base, or to sugar moietyanalogues, or to phosphate moiety analogues, or to base analogues.

[0111] All the described schemes demonstrate that BACs can not besynthesized in non-targeted cells because the molar concentration of thechemically active groups is too low, and without hybridization of theoligomer-PBACs to the template, specific reactions can not occur. Afterhybridization of the oligomer-PBACs to a specific template, theconcentration of the chemically active groups is sufficient for thechemical reaction between the chemical groups of PBACs to occur. Thereaction leads to chemical bond formation between PBACs and subsequentformation of a whole BAC. The degradation of the oligomers and/orlinking moieties of the oligomers with PBACs leads to the release ofBACS directly into targeted cells. To synthesise directly incellsbiologically active polymers such as proteins and RNAs ofdetermined structure more than two PBACs are used. PBACs for synthesisof proteins or RNAs are designated as PA_(n). PA_(n) are peptides oroligoribonucleotides. The mechanisms of the interaction of such PBACsare the same as in the synthesis of small biologically active compounds.The difference is that the PBACs (with the exception of the first andlast PBACs) are bound simultaneously to the 5′ and 3′ ends of theoligomers so that the direction of synthesis of the biologically activeprotein or RNA can be determined.

[0112] Possible functions of BACs synthesized by proposed methodsare: 1) Killing of cells, 2) Stimulation of the metabolism of cells 3)Blocking of important ion channels such as Na⁺, K⁺, Ca⁺⁺ and other ionchannels, in order to inhibit signal transmissions. BACs can beproteins, peptides, alkaloids, synthetic organic compounds. They can becleaved into two or more precursors called PBACs. After interactionbetween the chemical groups of PBACs, whole BAC is formed through themoiety “m”.

[0113] a) In Formula 1,2,3 and 4 PBACs are designated as “A” and

[0114] A-m-B is equal to a whole BAC “T”

[0115] “m” is selected independently from —S—S—, —O—, —NH—C(O)—,—C(O)—NH—, —C(O)—, —NH—, dbdN—, —C(O)O—, —C(O)S—, —S—, —C(S)S—, —C(S)O,—N═N—.

[0116] A-O-B is equal to a whole BAC “T”

[0117] A-NH—C(O)-B is equal to a whole BAC “T”

[0118] A-C(O)—NH-B is equal to a whole BAC “T”

[0119] A-C(O)-B is equal to a whole BAC “T”

[0120] A-NH-B is equal to a whole BAC “T”

[0121] A-dbdN-B is equal to a whole BAC “T”

[0122] A-C(O)O-B is equal to a whole BAC “T”

[0123] A-C(O)S-B is equal to a whole BAC “T”

[0124] A-C(S)S-B is equal to a whole BAC “T”

[0125] A-S—S-B is equal to a whole BAC “T”

[0126] A-C(S)O-B is equal to a whole BAC “T”

[0127] A-N═N-B is equal to a whole BAC “T”

[0128] b) A biologically active compound can be formed through themoieties “m” and “m^ 1”. “m” and “m^ 1” are selected independently from:—S—S—, —O—, —NH—C(O)—, —C(O)—NH—, —C(O)—, —NH—, dbdN—, —C(O)O—, —C(O)S—,—S—, —C(S)S—, —C(S)O—, —N═N—, so that

[0129] is equal to biologically active compound “T”

[0130] This kind of interaction is represented in FIG. 4.

[0131] c) In Formulas 5, 6 and 7, precursors of BACs (PBACS) aredesignated as “PA_(n)”, where n is selected from 2 to 2000. “PA” arepeptides or oligoribonucleotides consisting of from 2 to 100 aminoacids. n is the ordinal number of PA in a series of PAs and designatesthe sequence of binding of PAs to each other.

[0132] {“PA₁”-m-“PA₂”-m-“PA₃-m- . . .-m-“PA_(n-3)”-m-“PA_(n-2)”-m-“PA_(n-1)”-m-“PA_(n)”} is equal to BAC“PR”. BACs “PR” in this case are proteins or RNAs. Proteins can becellular proteins, enzymes, transcription factors, ligands, signallingproteins, transmembrane proteins, cytolitical toxins, cytoplasmic andnuclear proteins and the like. RNAs are selected from mRNA, rsRNA andthe like.

BRIEF DESCRIPTION OF DRAWNINGS

[0133]FIG. 1 Synthesis of the Toxin Daphnoretin.

[0134] Toxin Daphnoretin is cleaved into two precursors. Aftersimultaneous hybridization to cellular RNA of the oligomers bound to thedaphoretin's precursors, the chemically active hydroxyl group ofdaphnoretin's precursor “A” interacts with the chemically active Clgroup of precursor “B” to form a chemical bond between the twodaphnoretin precursors. The degradation of the linking moieties and/oroligomers leads to the release of the biologically active moleculedirectly into targeted cells.

[0135]FIG. 2 Synthesis of the Neurotoxin Peptide,

[0136] Neurotoxin is cleaved into two shorter, biologically inactivepeptides. After hybridization to cellular RNA or DNA, the chemicallyactive NH₂ group of peptide “A” interacts with the linking moiety—C(O)—O-L^ 2, forming a peptidyl bond. After the peptidyl bondsformation, the chemically active group —SH of peptide “B” interacts withthe linking moiety L^ 1-S—S— which binds peptide “A” with oligomer “A”.After this interaction, an —S—S— bound between the two cysteins isformed and the biologically active neurotoxin is released into targetedcells. Aminoacids are designated as italicised letters in one lettercode.

[0137]FIG. 3 The Synthesis of the Toxin Tulopsoid A.

[0138] Toxin tulopsoid A is cleaved into two precursors. Aftersimultaneous hybridization to cellular RNA of the oligomers bound to thetulopsoid A precursors chemically active hydroxyl group of theoligomer-PBAC “A” interacts with the —CH₂—S—C(O)— linking moiety to forma chemical bond with tulopsoid's precursor “B”, releasing precursor “B”from oligomer 2. The activated —CH₂—SH moiety interacts with the linkingmoiety —S—O—, releasing the whole tulopsoid A from oligomer 1.

[0139]FIG. 4 Synthesis of the Toxin Amanitin.

[0140] Toxin-amanitin is a strong inhibitor of transcription. It can becleaved into two inactive precursors which can be used to synthesise thewhole molecule of amanitin. After hybridization of all oligomers boundwith the amanitin's precursors to cellular RNA or DNA, free amino groupof amanitin's precursor “A” can interact with the carboxyl group—C(O)—S-L^ 2 to form a peptidyl bond and to release amanitin's precursor“B” from oligomer 2. The linking moiety of amanitin's precursor “A” tothe oligomer 1 is semistabile. The release of precursor “A” from theoligomer 1 is performed due to action of the activated —SH group on thelinking moiety —C(O)—O—S-L^ 1. Oligomers 3 and 4 bound with theamanitin's precursors “A” and “B” are hybridized on the same molecule ofRNA or DNA. The amino group of amanitin's precursor “B” interacts withthe carboxyl group —C(O)—S-L^ 1 to form a peptidyl bond, releasingamanitin's precursor “A” from the oligomer 3. The linking moiety ofamanitin's precursor “B” to the oligomer 4 is semistabile. The releaseof precursor “B” from the oligomer 4 is performed due to action of theactivated —SH group on the linking moiety —C(O)—O—S-L^ 2.

[0141]FIG. 5 Synthesis of the Toxin D-actinomicin.

[0142] Toxin D-actinomicin is cleaved into two precursors. Aftersimultaneous hybridization of two oligomer-PBACs to cellular RNA or DNAchemically active amino and halogen groups of precursor “A” interactwith the chemically active halogen and hydroxyl groups ofD-actinomicin's precursor “B” respectively to form two chemical bondsbetween the precursors.

[0143]FIG. 6 Synthesis of the Toxin Ochratoxin A.

[0144] Toxin ochratoxin A is cleaved into two precursors which are boundto oligomers. After simultaneous hybridization of the oligomcr-PBACs tocellular RNA or DNA, the chemically active amino group of precursor “B”interacts with the moiety C(O)—O— which links precursor “A” witholigomer A, to form a chemical bond between the two ochratoxinprecursors. After oligomer or linking moiety degradation in the cellsthe whole biologically active molecule of Ochratoxin A is released intothe targeted cells.

[0145]FIG. 7 Synthesis of the Toxin Ergotamin

[0146] Toxin ergotamin is cleaved into two precursors which are bound tooligomers. After simultaneous hybridization of the oligomer-PBACs tocellular RNA or DNA, the chemically active amino group of precursor “B”interacts with the moiety C(O)—O— which binds precursor “A” witholigomer “A”, to form a chemical bond between the two ergotaminprecursors. After degradation of the oligomers, RNA, or DNA in thecells, the whole biologically active molecule of ergotamin is releasedinto the targeted cells.

[0147]FIG. 8. Synthesis of Proteins.

[0148] The synthesis of a biologically active protein of n peptides.

[0149] Peptides are bound to oligomers simultaneously at their amino andcarboxy ends, with the exception of the first peptide which is bound tothe oligomer at its carboxy end, and the last peptide which is bound tothe oligomer at its amino terminus. Two oligomers bound to peptides(oligomer-PAs) are hybridized simultaneously to specific RNA or DNAmolecules, the distance from each other between 0 and 10 nucleotides ofcellular RNA or DNA. After hybridization, the amino group of theoligomer-PA_(n) interacts with the -L^ 2-S—C(O)— linking moiety to forma peptidyl bond between peptide “_(n-1)” and peptide “n”. Thepeptide_(n-1) is released from the oligomer_(n-1) at its carboxyterminus. The activated -L^ 2-SH group interacts then with the linkingmoieties —O—S-L^ 1 and —O—NH-L^ 1 which bind peptides_(n) at theirN-terminus with oligomers_(n). After hybridization of all n oligomer-PAsthe process is repeated n-1 times to bind all n peptides into onebiologically active protein. Linking of the peptides at the N-terminuswith oligomers is performed by aminoacids which have hydroxyl group suchas serine, threonine and tyrosine.

[0150]FIG. 9. Synthesis of Proteins.

[0151] The same process is shown as in FIG. 8, but this time thepeptides are bound at their N terminus to oligomers through aminoacidswith amino and-mercapto groups, for example cysteine, arginine,asparagine, glutamine and lysine. The activated -L^ 2-SH group caninteract with the linking groups such as —S—S-L^ 1, —S—NH-L^ 1 to form-L^ 2-S—S-L^ 1-, -L^ 2-S—NH-L^ 1 moieties and to release peptides fromoligomers at their N terminus.

[0152]FIG. 10. Synthesis of RNA

[0153] In this figure “PA_(n)” are oligoribonucleotides comprising from3 to 300 nucleotides.

[0154] n in “PA_(n)” means the ordinal number in a series ofoligoribonucleotides used in the synthesis of whole RNA, where n isselected from 2 to 1000.

[0155] PA₁ couples with PA₂ through the chemical moiety —O—, then inturn PA₁-m-PA₂ couples with PA₃ through chemical moiety —O—, thenPA₁-m-PA₂-m-PA₃ couples with PA₄ through chemical moiety —O— and so onuntil the last “n”th oligoribonucleotide is bound, forming the wholebiologically active RNA.

[0156] The chemical moieties in FIGS. from 1 to 10 are as follows:

[0157] m is selected independently from: —S—S—, —N(H)C(O)—, —C(O)N(H)—,—C(S)—O—, —C(S)—S—, —O—, —N═N—, —C(S)—, —C(O)—O—, —NH—, —S—;

[0158] K^ 1 is selected independently from: —NH(2), dbdNH, —OH, —SH, —S,—Cl, —Br, —I, —R^ 1-C(X)—X^ 1-R^ 2;

[0159] K^ 2 is selected independently from: —NH(2), -dbd-NH, —OH, —SH,—R^ 1-C(X)—X^ 1-R^ 2, —F, —Cl, —Br, —I;

[0160] L^ 1 is independently: chemical bond, —R^ 1-, —R^ 1-O—S—R^ 2-,—R^ 1-S—O—R^ 2-, —R^ 1-S—S—R^ 2-, —R^ 1-S—N(H)—R^ 2-, —R^ 1-N(H)—S—R^2-, —R^ 1-O—N(H)-—R^ 2-, —R^ 1-N(H)—O—R^ 2-, —R^ 1-C(X)—X—R^ 2-;

[0161] L^ 2 is independently: chemical bond, —R^ 1-, —R^ 1-O—S—R^ 2-,—R^ 1-S—O—R^ 2-, —R^ -S—S—R^ 2-, —R^ 1-S—N(H)—R^ 2-, —R^ 1-N(H)—S—R^ 2-,-R^ 1-O—N(H)—R^ 2-, —R^ 1-N(H)—O—R^ 2-, —R^ 1-C(X)—X^ 1-R^ 2-, —R^1-X—C(X)—X—C(X)—X—R^ 2-;

[0162] R^ 1 is independently: chemical bond, alkyl, alkenyl, alkynyl,aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl,cycloheteroaryl, carbocyclic, heterocyclic ring, X^ 1-P(X)(X)—X^ 1,—S(O)—, —S(O)(O)—, —X^ 1-S(X)(X)—X^ 1-, —C(O)—, —N(H)—, —N═N—, —X^1-P(X)(X)—X^ 1-, —X^ -P(X)(X)—X^ 1-P(X)(X)—X^ 1, —X^ 1-P(X)(X)—X^1-P(X)(X)—X^ 1-P(X)(X)—X^ 1, —C(S)—, any suitable linking group;

[0163] R2 is independently chemical bond, alkyl, alkenyl, alkynyl, aryl,heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloheteroaryl,carbocyclic, heterocyclic ring, X^ 1-P(X)(X)—X^ 1, —S(O)—, —S(O)(O)—,—X^ 1-S(X)(X)—X^ 1-, —C(O)—, —N(H)—, —N═N—, —X^ 1-P(X)(X)—X^ 1-, —X^1-P(X)(X)—X^ 1-P(X)(X)—X^ 1, —X^ 1-P(X)(X)—X^ 1-P(X)(X)—X^ 1-P(X)(X)—X^1, —C(S)—, any suitable linking group;

[0164] X is independently S, O, NH, Se, alkyl, alkenyl, alkynyl;

[0165] X^ 1 is independently S, O, NH, Se, alkyl, alkenyl, alkynyl.

BEST MODE FOR CARRYING OUT THE INVENTION The Synthesis of DifferentToxins and Alkaloids Directly Into Targeted Cells EXAMPLE 1 TheSynthesis of the Toxin Alpha Amanitin

[0166] The amanitin is a toxin present in mushrooms. It acts as a verystrong inhibitor of transcription in eucaryotic cells, and is thereforevery strong toxin.

[0167] The synthesis of alpha-amanitin is represented in FIG. 1 Thestructure of the toxin is a cyclic peptide with modified amino acids.The molecule of alpha-amanitin can be cleaved into two inactiveprecursors, which are bound to 4 oligomers through linking moieties L^ 1and L^ 2, designated in FIG. 1. After hybridization of all oligomers tothe same molecule of RNA the synthesis of toxin amanitin is occured.

EXAMPLE 2 The Synthesis of Biologically Active Peptides

[0168] The synthesis of BACs consisting of amino acids makes possiblethe synthesis of practically any peptide. These peptides can be involvedin a wide variety of processes. The specific synthesis will occur onlyin the cells where the specific sequences are represented.

[0169] The synthesis of peptides such as endorphins or toxins whichblock Na, K, Ca channels can be performed directly on specific RNA orDNA sequences. These peptides can act as agents stimulating cells of thenervous system, or as analgesic agents. To date, the number of knownbiologically active peptides is enormous. The peptides can besynthesized from natural aminoacids as well as from synthetic aminoacids of D or L conformations.

[0170] The synthesis of neurotoxin is represented in FIG. 2.

EXAMPLE 3 The Synthesis of the Toxin Tulopsoid A

[0171] Toxin tulopsoid A is an alkaloid and is a strong cytoliticaltoxin.

[0172] Toxin tulopsoid A is cleaved into two precursors. The chemicallyactive hydroxyl group of precursor “A” can interact after hybridizationwith the —CH₂—S—C(O)— moiety to form a chemical bond with tulopsoid'sprecursor “B”, with the release of precursor “B” from the oligomer. Theactivated —CH₂—SH moiety interacts with the linking moiety —S—O—,releasing the whole tulopsoid from oligomer (FIG. 3).

EXAMPLE 4 The Synthesis of the Toxin Daphnoretin

[0173] Toxin daphnoretin is an alkaloid and is a strong cytoliticaltoxin.

[0174] Toxin Daphnoretin is cleaved into two precursors. Aftersimultaneous hybridization of the oligomers coupled to the daphnoretin'sprecursors the chemically active hydroxyl group of daphnoretin'sprecursor “A” interacts with the chemically active Cl group of precursor“B” to form chemically bond between daphnoretin's precursors. Thedegradation of the oligomers or linking groups leads to the release ofthe biologically active molecule directly into targeted cells (FIG. 4).

EXAMPLE 5 The Synthesis of the Toxin D-actinomicin

[0175] Toxin D-actinomicin is an alkaloid and is a strong cytoliticaltoxin.

[0176] Toxin D-actinomicin is cleaved into two precursors. Afterhybridization of two oligomers to cellular RNA or DNA, the chemicallyactive groups amino and halogen of precursor “A” interact with thechemically active groups halogen and hydroxyl respectively ofD-actinomicin's precursor “B” to form two chemical bonds between theprecursors (FIG. 5.).

EXAMPLE 6 The Synthesis of the Toxin Ochratoxin A

[0177] Toxin ochratoxin A is an alkaloid and is a strong cytoliticaltoxin.

[0178] Toxin ochratoxin A is cleaved into two precursors bound tooligomers. After hybridization of the oligomers to cellular RNA or DNA,the chemically active amino group of the precursor “B” interacts withthe moiety —O—C(O) of precursor “A” to form a chemical bond between thetwo ochratoxin precursors. After the degradation of the oligomers orlinking moieties in the cells, whole, biologically active molecules ofOchratoxin A will be released into targeted cells (FIG. 6.).

EXAMPLE 7 The Synthesis of the Toxin Ergotamin

[0179] Toxin ergotamin is an alkaloid and is a strong cytolitical toxin.

[0180] Toxin ergotamin is cleaved into two precursors which are bound tooligomers. After hybridization of the oligomers to cellular RNA or DNA,the chemically active amino group of precursor “B” interacts with moiety—O—C(O) of precursor “A” to form a chemical bond between the twoergotamin precursors. After degradation of the oligomers or linkingmoieties in the cells, whole, biologically active molecules of ergotaminwill be released into the targeted cells.

[0181] By using more than two oligonucleotides bound at their 5′,3′ endsto precursors of biologically active compounds, higher concentrationlevel of the biologically active substances can be achieved intotargeted cells.

[0182] O11, O12, O13 are oligomers 1,2,3 which at their 3′ and 5′ endsare bound to precursors of biologically active substances.

[0183] Such linking can also prevent oligonucleotides from exonucleasedegradation and constabilise their activity in cells. In any case, theproducts of the degradation of the peptides and oligonucleotides formedfrom natural aminoacids and nucleotides are not toxic, and can be usedby cells without elimination from the organism or toxic effects on otherhealthy cells.

[0184] All the toxins described can be used for the synthesis of toxinsin cells infected by viruses, using the hybridization of the oligomersto double stranded DNA. In U.S. Pat. No. 5,571,937 the homopurinesequences of HIV 1 were found.

[0185] One such sequence is 5′-GAAGGAATAGAAGAAGAAGGTGGAGAGAGAGA-3′ (seqID NO 43 U.S. Pat. No. 5,571,937). Using two oligomers:(A-5′-GAAGGAATAGAAGAAG-3′) and (B-5′-AAGAAGGTGGAGAGAGAGA-3′) boundthrough linking moieties L^ 1 and L^ 2 to PBACs, synthesis of thecorresponding BACs directly in human cells infected by HIV1 can beachieved. The toxin will be synthesized only in thouse cells infected byHIV1. Other healthy cells will be not killed by synthesized toxin.

[0186] The Synthesis of Proteins

[0187] The synthesis of protein can be performed according to the schemedesignated in Formulas 5, 6 and 7 and in FIGS. 8, 9.

[0188] Relatively small molecules can be used to synthesize the wholeactive proteins in any tissue of a living organism. These smallmolecules can easily penetrate the blood brain barrier, or enter othertissues. The degradation products of such compounds can be used asnutrients for other cells. They are also not toxic to other cells wherespecific RNAs are not present, in the case where oligomers areoligoribo(deoxy)nucleotides. The synthesis of whole proteins of 50 kDacan be performed on one template 300-500 nucleotides in length usingoligomers of the length 10-50 nucleomonomers bound to peptidesconsisting of 2-30 amino acids. Only 10-20 such PBACs are necessary tosynthesise a protein of molecular weight 50 kDa. Theoretically, it ispossible to synthesise the proteins of any molecular mass. The number ofoligomer-PAs can vary from 1 to 1000, but the efficiency of synthesis oflarge proteins is very low and depends on the velocity of the reactionand the degradation of the oligomer-PAs in the living cells.

[0189] By this method, synthesized proteins can be modified later in thecells by cellular enzymes to achieve the biologically active form of theprotein.

[0190] The method allows the synthesis of specific proteins only inthouse cells in which the proteins are needed. Any type of proteins canbe synthesized by this method. These proteins can be involved incellular metabolism, transcription regulation, enzymatic reactions,translation regulation, cells division or apoptosis.

[0191] The mechanism allows the synthesis of any protein directly intotargeted cells. The synthesized proteins could inhibit a cell's growthor division, or could stimulate division and metabolism of cells wherespecific RNAs are expressed. By the method described, it is possible tosynthesise not only one protein, but many different proteins in theselected cells. These proteins could change even the differentiation ofthe targeted cells. The targeted cells can be somatic cells of livingorganisms, tumour cells, cells of different tissues, bacterial cells orcells infected by viruses.

EXAMPLE 8 Synthesis of the Tumour Suppresser p53

[0192] The synthesis is performed according to Formula 6. In the examplebelow, the peptides from PA₂ to PA₁₄ are bound at their NH₂ end to thelinking moiety L^ 2 through the OH group of amino acids serine orthreonine. The linking moiety L^ 2 is bound to the phosphate or sugarmoiety of the nucleotides localised at the 5′ end of the correspondingoligomers. The amino acids at the COOH ends of the peptides are bound tothe oligomer through acyl moieties (L^ 1) bound to the 3′ OH group ofsugar moiety of the nucleotide localised at 3′ end. After hybridizationto specific cellular RNA, the NH₂ group of the oligomer_(n)-PA_(n)interacts with the linking acyl group of the oligomer_(n-1)-PA_(n-1) toform a peptidyl bond between two oligomer-PAs. The whole P53 protein canbe synthesized using only 14 oligomer-PAs and a 250 nucleotide longregion of RNA for hybridization to the oligomer-PAs.

[0193] PA₁, PA₂, PA₃, PA₄, PA₅, PA₆, PA₇, PA₈, PA₉, PA₁₀, PA₁₁, PA₁₂,PA₁₃ and PA₁₄ are the peptides which are bound to the oligomers. Thesequences of the peptides are represented below.

[0194] PA₁—MEEPQSOPSV EPPLSQETFS DLWKLLPENN VL

[0195] PA₂—SPLPSQAM DDLMLSPDDI EQWF

[0196] PA₃—TEDPGPDEAP RMPEAAPRVA PAPAAP

[0197] PA₄—TPAAPAPAPS WPLSSSVPSQ KTYQG

[0198] PA₅—SYGFRLGFLHS GTAKSVTCRY

[0199] PA₆—SPAL NKMFCQLAKT CPVQLWVDSTPPPG

[0200] PA₇—TRVRAM AIYKQSQHMT EVVRRCPHHE

[0201] PA₈—TCSDSDGLAP PQHLIRVEGN LRVEYLDDRN

[0202] PA₉—TFRHSVVVPY EPPEVGSDCT TIHYNYMCNS

[0203] PA₁₀—SCMGGMNRRP ILTIITLEDS SGNLLGRN

[0204] PA₁₁—SFEVRVCACPGR DRRTEEENLR KKGEPHHELPPG

[0205] PA₁₂—STKRALPN NTSSSPQPKK KPLDGEYF

[0206] PA₁₃—TLQIRGRERFEM FRELNEALEL KDAQAGKEPGG

[0207] PA₁₄—SRAHSSHLK SKKGQSTSRH KKLMFKTEGP DSD

[0208] Aminoacids are designated in bold/italicised one letter code.

[0209] A—alanine, R—arginine, A—asparagine, D—aspartic acid, C—cysteine,Q—glutamine, E—glutamic acids, G—glycine, H—histidine, I—isoleucine,I—leucine, K—lysine, M—methionine, F—phenylalanine, P—proline, S—serine,T—threonine, W—tryptophan, Y—tyrosine, V—valine.

[0210] The tyrosine in PA₇ can be chemically phosphorylated. In this wayan already active form of the protein can be synthesized directly in thecells. It is possible to include any modification at any amino acid ofthe PAs. oligomer 1 5′-cccaatccctcttgcaactga-3′ oligomer 25′-attctactacaagtctgccctt-3′ oligomer 3 5′-ttgtgaccggctccactg-3′oligomer 4 5′-taccttggtacttctctaa-33′ oligomer 55′-atgccatattagcccatcaga-33′ oligomer 6 5′-ccaagcattctgtccctccttt-3′oligomer 7 5′-tccggtccggagcacca-3′ oligomer 8 5′-gccatgacctgtatgttaca-3′oligomer 9 5′-ggtgtgggaaagttagcggg-3′ oligomer 105′-gcgaattccaaatgattttaa-33′ oligomer 11 5′-aatgtgaacatgaataa-33′oligomer 12 5′-agagtgggatacagcatctata-3′ oligomer 135′-acaaaaccattccactctgatt-3′ oligomer 14 5′-ttggaaaaactgtgaaaaa-3′

[0211] All oligomers herein are oligonucleotides antiparallel to thehuman plasminogen antigen activator mRNA. After hybridization of theoligomer-PAs to the RNA, the distance between the 3′ ends of theoligomer_(n-1) and the 5′ ends of the oligomer_(n) is equal to 0nucleotides of plasminogen antigen activator mRNA. n as used herein isfrom 1 to 14.

[0212] H₂N-MEEPOSDPSVEPPLSQETFSDLWKLLPENNVL Oligomer1-PA₁ is                         L{circumflex over ( )}15′-cccaatcoctcttgcaactga-3′ H2N-SPLPSOAMDDLMLSPDDIEQWF Oligomer₂-PA₂ is  L{circumflex over ( ×2                 L{circumflex over ( )})}15′-attctactacaagtctgccctt-3′ H₂N-TEDPGPDEAPRMPEAAPRVAPAPMP Oligomer₃-PA₃is    L{circumflex over ( )}2            L{circumflex over ( )}15′-ttgtgaccggctccactg-3′ H₂N-TPAAPAPAPSWPLSSSVPSQKTYQG Oligomer₄-PA₄ is   L{circumflex over ( )}2                      L{circumflex over ( )}15′-taccttqgtacttctctaa-3′ H₂N-SYGFRLGFLHSGTAKSVTCTY Oligomer₅-PA₅ is   L{circumflex over ( )}2                 L{circumflex over ( )}15′-atgccatattagcccatcaga-3′ H₂N-SPALNKMFCQLAKTCPVQLWVDSTPPPGOligomer₆-PA₆ is    L{circumflex over( )}2                       L{circumflex over ( )}15′-ccaagcattctgtccctccttt-3′ H₂N-TRVRAMAIYKQSQHMTEVVRRCPHHEOligomer₇-PA₇ is    L{circumflex over( )}2                      L{circumflex over ( )}15′-tccggtccggagcacca-3′ H₂N-TCSDSDGLAPPQHLIRVEGNLRVEYLDDRN Oligomer₈-PA₈is   L{circumflex over ( )}2                          L{circumflex over( )}1 5′-gccatgacctgtatgttaca-3′ H₂N-TFRHSVVVPYEPPEVGSDCTTIHYNYMCNOligomer₉-PA₉ is    L{circumflex over( )}2                         L{circumflex over ( )}15′-ggtgtgggaaagttagcggg-3′ H₂N-SSCMGGMNRRPILTIITLEDSSGNLLGRNOligomer₁₀-PA₁₀ is    L{circumflex over( )}2                         L{circumflex over ( )}15′-gcgaattccaaatgattttaa-3′ H₂N-SFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGOligomer₁₁-PA₁₁ is  L{circumflex over( )}2                              L{circumflex over ( )}15′-aatgtgaacatgaataa-3′ H₂N-STKRALPNNTSSSPQPKKKPLDGEYF Oligomer₁₂-PA₁₂is      L{circumflex over ( )}2                    L{circumflex over( )}1 5′-agagtgggatacagcatctata-3′ H₂N-TLQIRGRERFEMFRELNEALELKDAQAGKEPGGOligomer₁₃-PA₁₃ is    L{circumflex over( )}2                             L{circumflex over ( )}15′-acaaaaccattccactctgatt-3′ H₂N-SRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSDOligomer₁₄-PA₁₄ is    L{circumflex over ( )}2 5′-ttggaaaaactgtgaaaaa-3′

[0213] The oligomer_(n)-PA_(n) (n is selected from 1 to 14) are peptideschemically bound to oligomers which can form stable duplex structurewith the plasminogen antigen activator mRNA expressed in human ovariantumour cells. Using the plasminogen antigen activator mRNA it ispossible to synthesize any other protein or small BAC. All theseproteins or BACs will be synthesized only in those cells where the humanplasminogen activator mRNA is expressed. In the case of the humanplasminogen activator mRNA, the synthesis of the protein or BAC willoccur only in ovarian tumour cells. Oligomer 1 at its 3′ end is bound tothe “C” end of the peptide PA₁ of p53 through the linking moiety L^ 1.Oligomers 2 to 13 are bound at their 5′ and 3′ ends to peptides PA₂ toPA₁₃ at their “N” and “C” ends respectively, through the linkingmoieties L^ 2 and L^ 1. Oligomer14 at it's 5′ end is bound to the “N”end of the peptide PA₁₄ of p53 through the linking moiety L^ 2. Thefirst methionine of PA₁ is formylated, and the amino end of peptide₁ isnot bound to Oligomer₁. The last amino acid at the carboxyl end of PA₁₄is not bound to Oligomer₁₄. Only 14 peptides chemically bound to 14oligomers are required to synthesize p53 tumour suppresser specificallyin the cells of the ovarian tumour. In any type of tumour cell RNAsspecific to this cell type are expressed. By this method, it is possibleto synthesise any protein or BACs described above on these RNAs.

[0214] The 14 Oligomer-PAs are hybridized on the mRNA in such a mannerthat the 3′ end of the oligomer₁-PA₁ is located at a distance from the5′ end of the oligomer₂-PA₂ which is equal to 0 nucleotides of theplasminogen antigen activator mRNA. The distance between the 5′ end ofthe Oligomer₃-PA₃ and the 3′ end of the Oligomer₂-PA₂ is equal to 0nucleotides of the plasminogen antigen activator mRNA. The distancebetween the 5′ end of the Oligomer₄-PA₄ and the 3′ end of theoligomer₃-PA₃ is equal to 0 nucleotides of the plasminogen antigenactivator mRNA etc. In other words, after hybridization of theoligomer-PAs to the plasminogen antigen activator mRNA, the distancebetween the 3′ end of the oligomer_(n-1)-PA_(n-1) and the 5′ end of theOligomer_(n)-PA_(n) is equal to 0 nucleotides of the plasminogen antigenactivator mRNA.

[0215] After the degradation of the oligomers and/or linking moieties,the synthesized protein p53 is released into the determined cells.

[0216]{H₂N-PA₂-C(O)NH-PA₂-C(O)NH-PA₃-C(O)NH-PA₄-C(O)NH-PA₅-C(O)NH-PA₆-C(O)NH-PA₇-C(O)NH-PA₈-C(O)NH-PA₉-C(O)NH-PA₁₀-C(O)NH-PA₁₁-C(O)NH-PA₁₂-C(O)NH-PA₁₃-C(O)NH-PA₁₄-COOH}is biologically active protein—tumour suppresser p53. The yield ofsynthesis in the cells can be very low, even <1%, because the synthesisoccurs directly in the targeted cells. Using different RNAs transcribedat different levels in the same cells, it is possible change the amountof the protein synthesized by this method.

[0217] The variety of proteins which can be synthesized by the proposedmethod is enormous. Limitations could occur if the proteins to besynthesised are very large or have many hydrophobic amino acids.

[0218] The distance between the 5′ and 3′ ends of the oligomer-PAs afterhybridization to the template can be varied between 0 and 10 nucleotidesof the target RNA.

[0219] In the example described above, the oligomers are antiparallel tothe plasminogen antigen activator mRNA. Using RNAs which expressedspecifically in different tumour cells, the synthesis of any protein inthese cells can be achieved. One example of such RNA is metastasin(mts-1) mRNA (Tulchinsky et al.1992, accession number g486654).

[0220] Using oligomers antiparallel to metastasin mRNA it is possible tosynthesise any toxin or protein specifically in human metastatic cells.

[0221] Using different RNAs expressed specifically in different tissuesor in cells infected by viruses, or in bacterial cells, it is possibleto synthesise any toxin or protein specifically in these cells.

THE EXAMPLE 10

[0222] Synthesis of the tumour suppresser p53 according to Formula 7.After hybridization of the oligomer-PAs to mRNA specific to ovariantumour cells (NbHOT Homo sapiens mRNA accession number AA402345), thechemical moiety K^ 1 of PA₂ (in this example K^ 1 is NH₂ group)interacts with the linking moiety L^ 2 of the oligomer₁-PA₁. After theinteraction has occurred, the peptide PA₁ is bound through the peptidylbond to the peptide PA₂ and is released from the 5′ end of theoligomer₁. The linking moiety L^ 2 of the oligomer₁ is activated so thatit interacts with the linking moiety L^ 1 of oligomer₂, and the peptidePA₁-C(O)NH-PA₂ is released from the 3′ end of oligomer₂. The chemicalmoiety K^ 1 of oligomer₃-PA₃ interacts with the linking moiety L^ 2 ofoligomer₂-{PA₁-C(O)NH-PA₂} to bind peptide PA₃ with PA₁-C(O)NH-PA₂,releasing peptide PA₁-C(O)NH-PA₂-C(O)NH-PA₃ from oligomer₂. Theactivated linking moiety L^ 2 of oligomer₂ interacts with the linkingmoiety L^ 1 and releases the peptide PA₁-C(O)NH-PA₂-C(O)NH-PA₃ from the3′ ends of oligomer₃. The processes described above are repeated in thecells 13 times. In such as manner, the protein:{PA₁-C(O)NH-PA₂-C(O)NH-PA₃-C(O)NH-PA₄-C(O)NH-PA₅-C(O)NH-PA₆-C(O)NH-PA₇-C(O)NH-PA₈-C(C)NH-PA₉-C(O)NH-PA₁₀-C(O)NH-PA₁₁-C(O)NH-PA₁₂-C(O)NH-PA₁₃-C(O)NH-PA₁₄}can be synthesized. Neither the degradation of the oligomers nor thedegradation of the linking moieties is necessary to release the proteinfrom the oligomers. Peptidyl bond formation between Pa_(n-1) and PA_(n)and degradation of the linking moieties L^ 2 proceed simultaneously withthe release of PAs from the 5′ ends of the oligomers. The activatedlinking moieties L^ 2 interact with the linking moieties L^ 1 to releasethe bound peptides from the 3′ ends of the oligomers.

[0223] PA₁—MEEPQSDPSVEPPLSOETFSDLWKLLPENNVL

[0224] PA₂—SPLPSQAMDDLMLSPDDIEQWF

[0225] PA₃—TEDPGPDEAPRMPEAAPRVAPAPAAP

[0226] PA₄—TPAAPAPAPSWPLSSSVPSQKTYQG

[0227] PA₅—SYGFRLGFLHSGTAKSVTCTY

[0228] PA₆—SPALNKMFCQLAKTCPVQLWVDSTPPPG

[0229] PA₇—TR VRAMAIYKQSQHMTEVVRRCPHHE

[0230] PA₈—TCSDSDGLAPPQHLIRVEGNLRVEYLDDRN

[0231] PA₉—TFRHSVVVPYEPPEVGSDCTTIHYNYMCNS

[0232] PA₁₀—SCMGGMNRRPIL TIITLEDSSGNLLGRN

[0233] PA₁₁—SFEVRVCACPGRORRTEEENLRKKGEPHHELPPG

[0234] PA₁₂—STKRALPNNTSSSPQPKKKPLDGEYF

[0235] PA₁₃—TL QIRGRERFEMFRELNEA LELKDA QA GKEPGG

[0236] PA₁₄—SRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD

[0237] where PA₁ to PA₁₄ are peptides bound to oligomers,

[0238] Aminoacids are designated in bold/italicised one letter code.

[0239] A—alanine, R—arginine, N—asparagine, D—aspartic acid, C—cysteine,Q—glutamine, E—glutamic acids, G—glycine, H—histidine, r—isoleucine,L—leucine, K—lysine, M—methionine, F—phenylalanine, P—proline, S—serine,T—threonine, W—tryptophan, Y—tyrosine, V—valine. Oligomer ₁3′ ATGGGCGGTAGGTAC 5′   Oligomer ₂ 3′ TAGCGGTGCCCTCGA 5′   Oligomer ₃3′ AACCCCGACGTCACG 5′   Oligomer ₄ 3′ TTCCGGACCCACGGA 5′   Oligomer ₅3′ CGAGGTACAGGCCCC 5′   Oligomer ₆ 3′ TACTCGAGTGTCTCG 5′   Oligomer ₇3′ ACGACCGTCCCTAGT 5′   Oligomer ₈ 3′ GACCGTGACTTCACC 5′   Oligomer ₉3′ TGACGGACGCCCGGA 5′   Oligomer ₁₀ 3′ CAGTCCTCGTCTAGC 5′   Oligomer ₁₁3′ TTCGACGTGAGTCCC 5′   Oligomer ₁₂ 3′ TCTCGGAGTCCCTTC 5′   Oligomer ₁₃3′ GGAGAGTCTGGTCGA 5′   Oligomer ₁₄ 3′ GGTCGGGTCGCGGGT 5′

[0240] Oligomers are complementary (antiparallel) to NbHOT Homo sapiensmRNA (clone 741045 accession number AA402345) which is specific toovarian tumour cells. The distance of the oligomers each from other isnull nucleotides of the NbHOT Homo sapiens mRNA.MEEPQSDPSVEPPLSQETFSDLWKLLPENNVL Oligomer₁-PA₁ is                                L{circumflex over ( )}23′ ATGGGCGGTAGGTAC 5′ (K{circumflex over ( )}1)SPLPSQAMDDLMLSPDDIEQWFOligomer₂-PA₂ is    L{circumflex over( )}1                     L{circumflex over ( )}2 3′ TAGCGGTGCCCTCGA 5′(K{circumflex over ( )}1)TEDPGPDEAPRMPEAAPRVAPAPAAP Oligomer₃-PA₃ is   L{circumflex over ( )}1                     L{circumflex over ( )}23′ AACCCCGACGTCACG 5′ (K{circumflex over ( )}1)TPAAPAPAPSWPLSSSVPSQKTYQGOligomer₄-PA₄ is    L{circumflex over( )}1                    L{circumflex over ( )}2 3′ TTCCGGACCCACGGA 5′(K{circumflex over ( )}1)SYGFRLGFLHSGTAKSVTCTY Oligomer₅-PA₅ is   L{circumflex over ( )}1                L{circumflex over ( )}23′ CGAGGTACAGGCCCC 5′ (K{circumflex over ( )}1)SPALNKMFCQLAKTCPVQLWVDSTPPPG Oligomer₆-PA₆ is    L{circumflex over( )}1                       L{circumflex over ( )}2 3 TACTCGAGTGTCTCG 5′(K{circumflex over ( )}1)TRVRAMAIYKOSQHMTEVVRRCPHHE Oligomer₇-PA₇ is   L{circumflex over ( )}1                     L{circumflex over ( )}23′ ACGACCGTCCCTAGT 5′ (K{circumflex over( )}1)TCSDSDGLAPPQHLIRVEGNLRVEYLDDRRN Oligomer₈-PA₈ is    L{circumflexover ( )}1                        L{circumflex over ( )}23′ GACCGTGACTTCACC 5′ (K{circumflex over( )}1)TFRHSVVVPYEPPEVGSDCTTIHYNYMCNS Oligomer₉-PA₉ is    L{circumflexover ( )}1                        L{circumflex over ( )}23′ TGACGGACGCCCGGA 5′ (K{circumflex over( )}1)SCMGGMNRRPILTIITLEDSSGNLLGRNS Oligomer₁₀-PA₁₀ is    L{circumflexover ( )}1                         L{circumflex over ( )}23′ CAGTCCTCGTCTAGC 5′ (K{circumflex over( )}1)FEVRVCACPGRDRRTEEENLRKKGEPHHELPPGS Oligomer₁₁-PA₁₁ is   L{circumflex over ( )}1                      L{circumflex over ( )}23′ TTCGACGTGAGTCCC 5′ (K{circumflex over ( )}1)TKRALPNNTSSSPQPKKKPLDGEYFOligomer₁₂-PA₁₂ is  L{circumflex over( )}1                       L{circumflex over ( )}2 3′ TCTCGGAGTCCCTTC5′({circumflex over ( )}1)TLQIRGRERFEMFRELNEALELKDAQAGKEPGGOligomer₁₃-PA₁₃ is   L{circumflex over( )}1                           L{circumflex over ( )}23′ GGAGAGTCTGGTCGA 5′ (K{circumflex over( )}1)SRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD Oligomer₁₄-PA₁₄ is   L{circumflex over ( )}1 3′ GGTCGGGTCGCGGGT 5′

[0241] This method of protein synthesis also allows modification of thesynthesized protein. Certain aminoacids of the peptides used in thesynthesis can be glycosylated or phosphorylated.

[0242] Glycosylation of a protein is a complex process, and difficultiesmay occur in the penetrance of some tissues with the glycosylated formof the peptide due to the size of the molecule.

[0243] However the use of phosphorylated peptides opens up thepossibility to synthesize already active proteins in the cells of livingorganisms.

[0244] The Synthesis of RNA.

[0245] Using the method described above, it is possible to synthesiseinto targeted cells not only proteins but also RNAs. An example of suchsynthesis is represented in FIG. 10

[0246] To synthesize whole RNA in cells from n oligomers bound tooligoribonucleotides (oligomer-PAs) the concentration of sucholigomer-PAs must be high. After the simultaneous hybridization ofoligomer-PAs to the same molecule of the cellular RNA, the chemicallyactive 3′ hydroxyl group of the oligoribonucleotid PA₁ interacts withthe linking moiety -L^ 2-S— which bound oligoribonucleotide PA₂ witholigomer 2. In this case the linking group is represented with a n —S-L^2-moiety which is coupled to phosphate group of the oligoribonucleotidePA₂. The 3′ hydroxyl group of the oligoribonucleotide PA₁ interacts withthe linking group of PA₂ forming a chemical bond with the phosphategroup, releasing the oligoribonucleotide PA₂ at it's 5′ end fromoligomer 2, and activating the linking moiety with the formation of the—SH group. This chemically active group —SH interacts with linkingmoiety -L^ 1-S which couples the oligomers. This process is repeated n-1times to bind all PAs in one molecule. PA₁ is bound through chemicalmoiety —O— to PA₂, then in turn PA₁-m-PA₂ is bound through chemicalmoiety —O— to PA₃, then PA₁-m-PA₂-m-PA₃ is bound through chemical moiety—O— to PA₄ and so on until the last oligoribonucleotide is bound,forming whole biologically active RNA.

[0247] In this figure “PA_(n)” are oligoribonucleotides comprising from3 to 300 nucleotides.

[0248] n in “PA_(n)” means the ordinal number in a series ofoligoribonucleotides used in the synthesis of a whole RNA, where n isselected from 2 to 1000.

1. A process for synthesis of biologically active compounds (BACs) frombiologically inactive BAC precursors (PBACs) “A”, “B” and “PA_(n)”chemically bound to 5′ and/or 3′ ends of the oligomers directly in cellsof living organisms according to Formulas 1 to 7, which processcomprises: (a) at least two oligomers, chemically bound at their 5′and/or 3′ ends to biologically inactive precursors of the biologicallyactive compounds (oligomer-PBACs), are hybridised simultaneously tocellular RNA, DNA or dsDNA in vivo in cells of a living organism, sothat after hybridization the distance between the 5′ or 3′ ends of theoligomer-PBAC “A” and the 3′ or 5′ ends of the oligomer-PBAC “B” is from0 to 8 ribo(deoxy)nucleotides of cellular RNA, DNA or dsDNAcorrespondingly, and the chemically active groups K^ 2 and K^ 1 of thebiologically inactive PBACs “A” and “B” can interact with each other orwith linking moieties L^ 1 and L^ 2 to form chemical moiety “m” betweenPBAC “A” and PBAC “B” so that “A”-m-“B” is equal to the biologicallyactive compound “T”; (b) (Formula 1) the same process as in (a), butafter hybridization of the “oligomer-PBACs” “A” and “B” to cellular RNA,DNA or dsDNA, the chemically active groups K^ 1 and K^ 2 of theoligomer-PBACs “A” and “B” interact with each other to form the chemicalmoiety “m”, which combines PBACs “A” and “B” into one active molecule ofthe biologically active compound “T”, the degradation of the oligomersand/or linking moieties L^ 1 and L^ 2 by cellular enzymes or hydrolysisleads to the release of the synthesized BAC “T” directly into targetedcells of a living organism; (c) (Formula 2) the same process as in (a),but after hybridization of “oligomer-PBACs” “A” and “B” to cellular RNA,DNA or dsDNA, the chemically active group K^ 2 of oligomer-PBAC “B”interacts with the linking moiety L^ 1 of oligomer-PBAC “A” to combinethe PBACs through chemical moiety “m”, into one active molecule of thebiologically active compound “T”, releasing the PBAC “B” from theoligomer and the oligomer “A” and/or linking moieties L^ 1 are degradedby cellular enzymes or hydrolysis leading to the release of thesynthesized BAC “T” directly into targeted cells of a living organism;(d) (Formula 3) the same process as in (a), but after hybridization of“oligomer-PBACs” “A” and “B” to cellular RNA, DNA or dsDNA, thechemically active group K^ 1 of the oligomer-PBACs interacts with thelinking moiety L^ 2 to combine the PBACs through chemical moiety “m”into one active molecule of the biologically active compound “T”,releasing the PBAC “B” from the oligomer and activating the chemicalmoiety L^ 2, which after activation interacts with the linking moiety L^1 to release the biologically active compound “T” from oligomer directlyinto targeted cells of a living organism. (e) (Formula 4) the sameprocess as in (a), but after hybridization of “oligomer-PBACs” “A” and“B” to cellular RNA, DNA or dsDNA, the chemically active group K^ 2 ofoligomer-PBAC “B” interacts with the linking moiety L^ 1 of theoligomer-PBAC “A” to combine the PBACs through the chemical moiety “m”,and the chemically active group K^ 1 of the oligomer-PBAC “A” interactswith the linking moiety L^ 2 of the oligomer-PBAC “B” to form chemicalmoiety m^ 1 which, together with the chemical moiety m, combines two“PBACs” into one active molecule of the biologically active compound“T”, with the release of the PBAC “B” from the oligomer.
 2. The processof claim 1 but: (a) the synthesis of the BAC “PR” in the cells of livingorganisms is performed from n “oligomer_(n)-PA_(n)”s so that“oligomer_(n-1)-PA_(n-1)” and “oligomer_(n)-PA_(n)” are hybridizedsimultaneously on the same molecule of cellular RNA, DNA or dsDNA, witha distance of from null to eight nucleotides of cellular RNA or DNAbetween the 3′ or 5′ ends of the oligomer_(n-1)-“PA_(n-1)”, and the 5′or 3′ ends of the oligomer_(n)-“PA_(n)” correspondingly, here n isselected from 2 to 2000; (b) (Formula 5) the same process as in (a), butafter simultaneous hybridization of “oligomer_(n-1)-PA_(n-1)” and“oligomer_(n)-PA_(n)” to cellular RNA or DNA, the chemically activegroups K^ 1 and K^ 2 interact with each other to form the chemicalmoiety “m” between “oligomer_(n-1)-PA_(n-1)” and “oligomer_(n)-PA_(n)”correspondingly, this step is repeated in the cells n-1 times andcombines n-1 times all “PA_(n)”s into one active molecule ofbiologically active compound “PR” which consists of n PA_(n) so that thecompound {“PA”₁-m-“PA”₂-m-“PA”₃-m-“PA”₄-m- . . .-m-“PA_(n-3)”-m-“PA_(n-2)”-m-“PA_(n-1)”-m-“PA_(n)”} is the biologicallyactive compound “PR”; the degradation of the oligomers and/or linkingmoieties L^ 1 and L^ 2 leads to the release of synthesized BAC “PR”directly in the targeted cells of a living organism, here n is selectedfrom 2 to 2000; (c) (Formula 6) the same process as in (a), but aftersimultaneous hybridization of “oligomer_(n-1)-PA_(n-1)” and“oligomer_(n)-PA_(n)” to cellular RNA, DNA or dsDNA chemically activegroup K^ 1 of “oligomer_(n-1)-PA_(n-1)” interacts with the linkingmoiety L^ 2 of “oligomer_(n)-PA_(n)” to bind PA_(n-1) and PA_(n) throughthe chemical moiety “m”, this step is repeated in the cells n-1 times,and combines n-1 times all PA_(n)s after hybridization of all n“Oligomer_(n)-PA_(n)”s into one active molecule of biologically activecompound “PR”, which consists of n PA_(n) so that the compound{PA₁-m-PA₂-m-PA₃-m-PA₄-m- . . .-m-PA_(n-3)-m-PA_(n-2)-m-PA_(n-1)-m-PA_(n)} is equal to the biologicallyactive compound PR; the degradation of the oligomers and/or linkingmoieties L^ 1 and L^ 2 due to cellular enzymes or hydrolysis leads tothe release of the synthesized BAC “PR” directly into targeted cells ofa living organism, here n is selected from 2 to 2000; (d) (Formula 7)the same process as in (c), but after interaction of K^ 1 with L^ 2, L^2 is chemically activated so that it can interact with the linkingmoiety L^ 1 of oligomer-PA_(n-1), destroying the binding ofoligomer_(n-1) with PA_(n-1), this step is repeated n-1 times, so thatonly whole BAC “PR” consisting of n PA_(n)s (PA₁-m-PA₂-m-PA₃-m PA₄-m- .. . -m-PA_(n-3)-m-PA_(n-2)-m-PA_(n-1)-m-PA_(n)) is released directlyinto targeted cells of a living organism, here n is selected from 2 to2000.
 3. In claims 1 and 2 the linking moieties L^ 1 and L^ 2 are boundto the first and/or last mononucleomers of the oligomers at their sugaror phosphate moiety, or directly to base, or to sugar moiety analogues,or to phosphate moiety analogues, or to base analogues.
 4. In claim 1,biologically inactive precursors of BAC “A” and “B” are selected fromchemical substances which can be bound to each other through thechemical moiety “m”, so that the compound A-m-B is the biologicallyactive compound “T”: A-O-B is equal to a whole BAC “T” A-NH—C(O)-B isequal to a whole BAC “T” A-C(O)—NH-B is equal to a whole BAC “T”A-C(O)-B is equal to a whole BAC “T” A-C(S)-B is equal to a whole BAC“T” A-NH-B is equal to a whole BAC “T” A-dbdN-B is equal to a whole BAC“T” A-C(O)O-B is equal to a whole BAC “T” A-C(O)S-B is equal to a wholeBAC “T” A-C(S)S-B is equal to a whole BAC “T” A-S—S-B is equal to awhole BAC “T” A-C(S)O-B is equal to a whole BAC “T” A-N═N-B is equal toa whole BAC “T”
 5. In claim 2, biologically inactive precursors of BACPA_(n) are selected from biologically inactive peptides andoligoribonucleotides so that the compound {“PA₁”-m-“PA₂”-m-“PA₃”-m- . .. -m-“PA_(n-2)”-m-“PA_(n-1)”-m-“PA_(n)”} is equal to the biologicallyactive compound “PR”, which is a protein or a RNA.
 6. Chemical moietiesin claims 1, 2, 3 and 4 are as follows: m is selected independentlyfrom: —S—S—, —N(H)C(O)—, —C(O)N(H)—, —C(S)—O—, —C(S)—S—, —O—, —N═N—,—C(S)—, —C(O)—O—, —NH—, —S—; K^ 1 is selected independently from:—NH(2), dbdNH, —OH, —SH, —F, —Cl, —Br, —I, —R^ 1-C(X)—X^ 1-R^ 2; K^ 2 isselected independently from: —NH(2), -dbd-NH, —OH, —SH, —R^ 1-C(X)—X^1-R^ 2, —F, —Cl, —Br, —I; L^ 1 is independently: chemical bond, —R^ 1-,—R^ 1-O—S—R^ 2-, —R^ 1-S—O—R^ 2-, —R^ 1-S—S—R^ 2-, —R^ 1-S—N(H)—R₂-, —R^1-N(H)—S—R^ 2-, —R^ 1-O—N(H)—R^ 2-, —R^ 1-N(H)—O—R^ 2-, —R^ 1-C(X)—X—R^2-; L^ 2 is independently: chemical bond, —R^ 1-, —R^ 1-O—S—R^ 2-, —R^1-S—C—R^ 2-, —R^ 1-S—S—R^ 2-, —R^ 1-S—N(H)—R^ 2-, —R^ 1-N(H)—S—R^ 2-,—R^ 1-O—N(H)—R^ 2-, —R^ 1-N(H)—O—R^ 2-, —R^ 1-C(X)—X^ 1-R^ 2-, —R^1-X—C(X)—X—C(X)—X—R ^ 2-; R^ 1 is independently: chemical bond, alkyl,alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl,heteroaryl, cycloheteroaryl, carbocyclic, heterocyclic ring, X^1-P(X)(X)—X^ 1, —S(O)—, —S(O)(O)—, —X^ 1-S(X)(X)—X ^ 1-, —C(O)—, —N(H)—,—N═N—, —X^ 1-P(X)(X)—X^ 1-, —X^ 1-P(X)(X)—X^ 1-P(X)(X)—X^ 1, —X^1-P(X)(X)—X^ 1-P(X)(X)—X^ 1-P(X)(X)—X^ 1, —C(S)—, any suitable linkinggroup; R^ 2 is independently chemical bond, alkyl, alkenyl, alkynyl,aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl,cycloheteroaryl, carbocyclic, heterocyclic ring, X^ 1-P(X)(X)—X^ 1,—S(O)—, —S(O)(O)—, —X^ 1-S(X)(X)—X^ 1-, —C(O)—, —N(H)—, —N═N—, —X^1-P(X)(X)—X^ 1-, —X^ 1-P(X)(X)—X^ 1-P(X)(X)—X^ 1, —X^ 1-P(X)(X)—X^1-P(X)(X)—X^ 1-P(X)(X)—X^ 1, —C(S)—, any suitable linking group; X isindependently S, O, NH, Se, alkyl, alkenyl, alkynyl; X^ 1 isindependently S, O, NH, Se, alkyl, alkenyl, alkynyl.
 7. Biologicallyactive compound “T” which can be synthesized according the processespresented in claims 1 and 3 include but are not limited to: a)biologically active alkaloids and their chemical analogues, peptides andinhibitors or cofactors of cellular enzymes; b) synthetic and naturalcompounds which are inhibitors or stimulators of cellular processes suchas: cellular metabolism, DNA replication, RNA transcription, RNAtranslation, RNA elongation and RNA processing, protein synthesis,protein processing, cellular differentiation, cellular division, ionchannel transmission, cellular protein and RNA's transportation,processes of cellular oxidation and the like.
 8. Biologically activecompounds “T” and “PR” in claims 1, 2, 3 and 4 include but are notlimited to cytolitical toxins and toxins.
 9. Biologically activecompounds “PR” which are synthesized according to the processespresented in claims 2 and 4 are selected from biologically activeproteins and RNAs.
 10. The biologically active proteins and peptidesdescribed in claims 2, 4 and 8 are synthesized from shorter biologicallyinactive peptides (PAs) consisting of from 2 to,100 aminoacids and theirsynthetic analogues L, D or DL configuration at the alpha carbon atomwhich are selected from valine, leucine, alanine, glycine, tyrosine,tryptophan, tryptophan isoleucine, proline, histidine, lysin, glutamicacid, methionine, serine, cysteine, glutamine phenylalanine, methioninesulfoxide, threonine, arginine, aspartic acid, asparagin, phenylglycine,norleucine, norvaline, alpha-aminobutyric acid, O-methylserine,O-ethylserine, S-methylcysteine, S-benzylcysteine, S-ethylcysteine,5,5,5-trifluoroleucine and hexafluoroleucine; other modifications ofaminoacids are also possible, including but not limited to the additionof substituents at carbone atoms such as —OH, —SH, —SCH₃, —OCH₃, —F,—Cl, —Br, —NH₂, —C(S)— or —C(O)—.
 11. The biologically active proteinsdescribed in claims 8 and 9 include but are not limited to enzymes, DNApolymerases, RNA polymerases, esterases, lipases, proteases, kinases,transferases, transcription factors, transmembrane proteins, membraneproteins, cyclins, cytoplasmic proteins, nuclear proteins, toxins andlike this.
 12. The biologically active RNAs described in Formula 2 canbe synthesized from biologically inactive oligoribonucleotidesconsisting of from 2 to 100 ribonucleotides, selected from uridin,guanidine, cytosin or adenine.
 13. In claims 1 and 2, the cells wherethe biologically active substances can be synthesized have specific RNA,DNA or dsDNA molecules of determined sequence.